De Candia E, De Cristofaro R, De Marco L, Mazzucato M, Picozzi M, Landolfi R
Centro Ricerche Fisiopatologia dell'Emostasi, Istituto di Semeiotica Medica, Università Cattolica S. Cuore, Roma, Italia.
Thromb Haemost. 1997 Apr;77(4):735-40.
The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.
血小板膜糖蛋白Ib（GpIb）具有与α-凝血酶的高亲和力结合位点，其占据被认为可正向调节凝血酶诱导的血小板活化。在本研究中，为了进一步表征凝血酶与GpIb的相互作用，研究了两个被称为“肝素结合位点”（HBS）和“纤维蛋白原识别位点”（FRS）的凝血酶阴离子外位点，作为可能参与GpIb结合的结构域。通过在肝素存在下以及对凝血酶肝素结合位点进行化学修饰（凝血酶-HBS磷酸吡哆醛化）后，进行125I-α-凝血酶与纯化的糖萼素（GC，GpIb的胞外部分）的结合测量，探讨了凝血酶HBS的作用。这些研究表明：a）肝素可竞争性抑制凝血酶与GC的结合；b）通过对HBS进行化学修饰，凝血酶与GC相互作用的平衡缔合常数降低了多达10倍。另一方面，FRS在凝血酶与GC相互作用中的作用可通过其他实验排除，这些实验表明溶液中的GC不会影响α-凝血酶与两种结合至催化位点和纤维蛋白原识别位点的底物的相互作用：1）凝血酶受体肽38-60（TR，L38-E60）和2）纤维蛋白原的Aα链。总之，这些结果表明GC与凝血酶的相互作用涉及酶的肝素结合位点，而纤维蛋白原识别位点不起重要作用。
